The present invention relates to a method of determining an amount of hemoglobin (Hb) in a sample.
Hb in the blood plays an important role in transporting oxygen from the lungs to organs and thus relates to diseases such as leukemia, anemia, and the like, for example. Therefore, determining an amount of Hb has been considered very important in the field of a clinical analysis. On the other hand, glycated Hb serves as an important index for the diagnosis, treatment, etc. of diabetes because it reflects previous blood glucose levels in vivo. Therefore, determining a ratio of glycated Hb also has been considered important. For determining the ratio of glycated Hb, it is necessary to determine the amount of Hb.
Examples of a method of determining Hb include measuring an absorbance of Hb. However, the Hb that is not yet denatured (hereinafter, referred to as xe2x80x9cundenatured Hbxe2x80x9d) exhibits an absorption maximum at different wavelengths depending on its state, e.g., the state where it is bound to oxygen, the state where it is not bound to oxygen, etc. Therefore, it is difficult to determine an amount of Hb accurately by merely measuring the absorbance of the Hb. On this account, conventionally, a method has been employed in which the absorbance of Hb is measured after the Hb has been denatured so as to be stabilized. Examples of such a method include a cyanmethemoglobin method (HiCN method), azide metohemoglobin method, sodium lauryl sulfate method (SLS method), alkaline hematin method, and the like. Among these, the HiCN method, which is an international standard method, is employed particularly widely. In this HiCN method, a reagent containing potassium ferricyanide and potassium cyanide is added to blood so that Hb is converted into stable cyanmethemoglobin, and the absorbance is measured at a predetermined wavelength (540 nm) to determine the amount of the Hb.
However, the HiCN method and the azide metohemoglobin method are not preferable from the viewpoint of environmental friendliness because the HiCN method produces a toxic liquid waste containing a cyanogen compound and the azide metohemoglobin method produces a liquid waste containing sodium azide. Further, the SLS method and the alkaline hematin method have the following problem. In these methods, a reagent such as SLS and a strong alkali is added to a sample as a protein-denaturing agent. Thus, when the sample containing such a reagent is used directly for the determination of a substance other than Hb, the reagent affects the system of determination utilizing an enzyme and the like, for example, which makes the determination difficult. Therefore, with respect to the sample that has been subjected to the treatment for denaturing Hb, it is difficult to carry out the determination of the denatured Hb and the determination of the substance other than the denatured Hb in a series of operations.
Therefore, it is an object of the present invention to provide a method of determining Hb, by which an amount of Hb can be determined easily and accurately without fear of damage to the environment.
In order to achieve the above object, a method of determining Hb according to the present invention includes: denaturing Hb in a sample with a tetrazolium compound to give denatured Hb; measuring an amount of an optical change in the sample at an absorption wavelength specific to the denatured Hb; and calculating an amount of the Hb in the sample from the amount of the optical change. The term xe2x80x9cdenatured Hbxe2x80x9d as used in the present invention refers to Hb that has been denatured with a tetrazolium compound.
While the undenatured Hb exhibits various absorption wavelengths depending on its state, the denatured Hb obtained by the treatment with the tetrazolium compound is stable and exhibits the absorption maximum at a wavelength falling within a certain range. Therefore, according to the method of determining Hb of the present invention, an amount of Hb can be determined easily. In addition, the method of the present invention does not use a cyanogen compound, a strong alkali, or the like as used in the above-mentioned conventional methods. Therefore, the method of the present invention is a useful method without fear of damage to the environment.
In the method of determining Hb according to the present invention, the amount of the optical change may be an absorbance, reflectance, or the like, for example.
The tetrazolium compound is not specifically limited. For example, tetrazolium compounds described later can be used. Among these, 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium salt (e.g., available from Dojindo Laboratories under the trade name WST-3) is most preferable.
In the method of determining Hb according to the present invention, the wavelength for measuring the denatured Hb preferably is in the range from 440 to 700 nm, more preferably from 500 to 670 nm, and most preferably 540 to 670 nm.
In the method of determining Hb according to the present invention, the amount of the tetrazolium compound added to the sample is not specifically limited and can be decided as appropriate depending on the type of the sample and the like. More specifically, it is preferable that the tetrazolium compound is added to the sample so that a content of the tetrazolium compound per microliter of the sample is in the range from 0.001 to 100 xcexcmol, more preferably from 0.01 to 10 xcexcmol, and most preferably from 0.05 to 5 xcexcmol, for example.
The sample is not specifically limited. However, as described later, a sample containing red blood cells, e.g., whole blood, preferably is used. In the case where the sample is whole blood, the tetrazolium compound preferably is added to the sample so that a content of the tetrazolium compound per microliter of the sample is in the range from 0.01 to 30 xcexcmol, more preferably from 0.05 to 10 mol, and most preferably from 0.1 to 5 xcexcmol, for example. In general, it is estimated that whole blood contains about 50 vol % of blood cells.
In the method of determining Hb according to the present invention, it is preferable that the Hb in the sample is treated with the tetrazolium compound in the presence of a surfactant. When the surfactant is present in addition to the tetrazolium compound, denaturation of the Hb can be accelerated still further so that the Hb can be determined quickly.
In the method of determining Hb according to the present invention, the amount of the surfactant added to the sample is not specifically limited, and can be decided as appropriate, for example, depending on the amount of the tetrazolium compound added to the sample and the like. More specifically, the surfactant is added to the sample so that a content of the surfactant per mole of the tetrazolium compound is in the range from 0.01 to 70 mol, preferably from 0.05 to 50 mol, and more preferably from 0.1 to 20 mol.
In the method of determining Hb according to the present invention, the sample is not specifically limited and can be, for example, blood samples such as whole blood, plasma, serum, blood cells, and the like. It is preferable to use a sample containing red blood cells, for example, a whole blood sample, a blood cell sample, etc.
Next, a method of determining a ratio of glycated Hb according to the present invention includes: determining an amount of Hb in a sample containing glycated Hb by the method of determining Hb according to the present invention; causing a redox reaction between a glycation site of the denatured Hb obtained and a fructosyl amino acid oxidase (hereinafter, referred to as xe2x80x9cFAODxe2x80x9d); determining the redox reaction to determine an amount of the glycated Hb; and calculating a ratio of the glycated Hb from the amount of the Hb and the amount of the glycated Hb. It is to be noted here that the amount of Hb refers to the amount of both the glycated Hb and the Hb that is not glycated.
Similarly to the method of determining Hb described above, the method of determining a ratio of glycated Hb according to the present invention also produces no toxic liquid wastes. Therefore, the method of determining a ratio of glycated Hb according to the present invention also is a useful method without fear of damage to the environment. In addition, unlike the strongly alkaline reagent and the like as described above, the tetrazolium compound does not have any effect, such as deactivation of an enzyme, on the determination of the amount of glycated Hb by the enzymic method utilizing the redox reaction. Accordingly, the determination of the Hb as described above and the determination of the amount of the glycated Hb can be carried out simply in a series of operations using the sample treated with the tetrazolium compound. Therefore, the determination of the ratio of glycated Hb according to the present invention can be carried out, for example, utilizing only one reaction system using a single measuring apparatus. Moreover, the tetrazolium compound added to the sample not only can denature the Hb but also can eliminate the effect of a reducing substance present in the sample on the redox reaction. Therefore, the ratio of glycated Hb can be determined with higher accuracy.
In the method of determining a ratio of glycated Hb according to the present invention, the wavelength for measuring the denatured Hb preferably is in the range from 440 to 700 nm, more preferably from 500 to 670 nm, and most preferably 540 to 670 nm as described above.
In the method of determining a ratio of glycated Hb according to the present invention, it is preferable that determining the redox reaction is measuring an amount of an optical change in a color-developing substance produced by the redox reaction. As described above, the amount of the optical change may be an absorbance, reflectance, or the like, for example.
In the enzymic method as described above, in general, dual-wavelength measurement dominantly is used for measuring the amount of the optical change. The dual-wavelength measurement is carried out, for example, in the following manner. First, an absorbance of a substance to be measured (e.g., a color-developing substance) is measured at a main wavelength at which the substance exhibits an absorption maximum. Then, at a sub-wavelength that is different from the main wavelength, electrical noises, cloudiness of the sample, changes in the amount of light, etc. are measured to correct the measured value obtained at the main wavelength. Therefore, it is preferable that the sub-wavelength is a wavelength at which a substance other than the substance to be measured (the color-developing substance) present in the sample exhibits no absorption. The main wavelength is decided as appropriate depending on the absorption wavelength of the substance to be measured. In general, the main wavelength is in the range from about 650 to 800 nm. In this case, the sub-wavelength is set to be higher than the main wavelength, e.g., in the range from about 800 to 900 nm. However, as described above, the undenatured Hb is unstable and exhibits absorption at various wavelengths depending on its state, e.g., the state where it is bound to oxygen, the state where it is not bound to oxygen, etc. Thus, in the case where the amount of the glycated Hb is determined, for example, by measuring the absorbance of the color-developing substrate and the like, the Hb exhibits absorption also at the sub-wavelength, which makes it difficult to determine the amount of the glycated Hb accurately. In contrast, the wavelength for measuring the denatured Hb obtained by the treatment with the tetrazolium compound is as described above. Therefore, even when the sub-wavelength in the determination of the amount of glycated Hb is set in the range from 800 nm to 900 nm, for example, the effect of the absorption by the unstable undenatured Hb, which exhibits absorption at various wavelengths depending on its state, is not observed. As a result, the amount of the glycated Hb can be determined with higher accuracy.
A wavelength for measuring the color-developing substance is in the range from 500 to 800 nm, preferably from 540 to 750 nm, for example.
Further, as described later, a wavelength for measuring the color-developing substance may be the same as that for measuring the amount of the Hb. In this case, the wavelength preferably is in the range from 500 to 670 nm, more preferably from 540 to 670 nm.
In the method of determining a ratio of glycated Hb according to the present invention, it is preferable that the denatured Hb is treated with a protease before causing the redox reaction between the glycation site of the denatured Hb and the FAOD. The FAOD acts on a glycated amino acid and a glycated peptide fragment shorter than a glycated protein and a glycated peptide more easily than on the glycated protein and the glycated peptide. On this account, the denatured Hb is degraded with a protease in advance so that the FAOD can act on the glycation site of the denatured Hb more easily, thereby allowing the accuracy of determination to be improved.
As described above, by using the method of determining Hb according to the present invention, it becomes possible to use the sample that has been subjected to the treatment for denaturing Hb directly for the determination of the amount of the glycated Hb. Therefore, the determination of the amount of the Hb and the determination of the amount of the glycated Hb can be carried out, for example, in the following orders.
For example, a first method of determining a ratio of glycated Hb is a method in which the redox reaction is caused between the glycation site of the denatured Hb and the FAOD after the amount of the optical change in the sample is measured at the absorption wavelength specific to the denatured Hb.
In the case where a protease treatment is carried out in this first method, the method may be carried out in such a manner that the amount of the optical change in the sample is measured at the absorption wavelength specific to the denatured Hb, the denatured Hb is then treated with a protease, and thereafter, a redox reaction is caused between a glycation site of a degradation product of the denatured Hb and FAOD. Alternatively, the method may be carried out in such a manner that the denatured Hb is treated with a protease, the amount of the optical change in the sample is then measured at the absorption wavelength specific to the denatured Hb, and thereafter, a redox reaction is caused between a glycation site of a degradation product of the denatured Hb and FAOD.
On the other hand, for example, a second method of determining a ratio of glycated Hb is a method in which, after the redox reaction is caused between the glycation site of the denatured Hb and the FAOD, the amount of the optical change in the sample is measured at the absorption wavelength specific to the denatured Hb and the redox reaction is determined. In the case where the second method is employed, by measuring the denatured Hb and the color-developing substance at the wavelengths different from each other, it becomes possible to determine the amount of the optical change in both the denatured Hb and the color-developing substance at the same time.
In the case where a protease treatment is carried out in this second method, the denatured Hb may be treated with a protease before the redox reaction is caused between the glycation site of the denatured Hb and the FAOD, for example.
In the method of determining a ratio of glycated Hb according to the present invention, it is preferable that the amount of the optical change in the color-developing substance corresponds to an amount of hydrogen peroxide generated by the redox reaction between the glycation site of the denatured Hb and the FAOD. Further, it is preferable that the color-developing substance is a substrate that develops color by oxidation (hereinafter, referred to as a color developing substrate) and has developed color as a result of a reaction caused by an oxidase between the hydrogen peroxide and the substrate.
The oxidase is not specifically limited. However, POD preferably is used as the oxidase. Also, the color-developing substrate is not specifically limited. However, N-(carboxymethylaminocarbonyl)-4,4xe2x80x2-bis(dimethylamino) diphenylamine sodium (e.g., available from Wako Pure Chemical Industries, Ltd. under the trade name DA-64), for example, preferably is used as the color-developing substrate because it can be detected with high sensitivity.
The sample used in the method of determining a ratio of glycated Hb according to the present invention is not specifically limited. Examples of the sample include those described above.